Functional and structural studies of protein inhibitors of RNase E activity that globally modulate mRNA abundance in Escherichia coli

Considerable progress has been made during recent years on how the regulation of mRNA abundance affects the level of gene expression. The 118 kDa protein ribonuclease E (RNase E), which was discovered initially by its ability to process E. coli 9S RNA, is now known to have a central role in mRNA decay, the maturation of tRNA, and the degradation of small regulatory RNA. RNase E contains two functionally distinct parts: the N-terminal half (NTH) which is responsible for the catalytic function and the C-terminal half (CTH) which provides a scaffold for the interaction with multiple proteins. Genetic analysis in our lab led to the discovery of RraA and RraB, two transacting protein inhibitors of the E. coli RNase E whose overexpression affects the stability of a large number of transcripts. The work presented here describes the functional and structural characterization of RraA and RraB. The 17.4 kDa protein RraA and the 15.6 kDa protein RraB inhibit the endoribonucleolytic activity of RNase E in vivo and in vitro. Co-precipitation experiments and SPR analysis using BIACore showed that both inhibitors physically interact with RNase E with a similar affinity. However, coprecipitation analysis using a set of RNase E mutants containing deletions within the scaffold region of RNase revealed that RraA and RraB bind to different sites on the enzyme. Specifically, RraA may have one or more weak binding sites on the NTH of RNase, but it requires the presence of CTH for high affinity interaction. In contrast, only a short region in the CTH, comprising of residues 694-727, is important for the binding of RraB. Microarray analysis also demonstrated that RraA affects the abundance of a distinct set of mRNAs which only partially overlaps with that of RraB . The crystal structure of the E. coli RraA showed that RraA crystallizes as a homotrimer and forms a ring-like structure with a center hole which is approximately 12 Å across. As part of this study we crystallized the Vibrio cholerae homolog of RraB (VC0424). Crystals were shown to diffract to about 3 Å but unfortunately a high resolution model of the structure of the protein could not be determined.